Cells for biochemical analysis, kit for biochemical analysis, and biochemical analyzer

ABSTRACT

The invention makes it possible to measure binding of a biochemical substance with a high throughput and with high sensitivity using a small cell capable of being filled with a small amount of chemical solution. A space between a first substrate and a second substrate such that probes are immobilized on their mutually facing planes is used as a cell that houses a specimen solution. Light is irradiated from a first substrate side, and reflected light is subjected to spectroscopy. Binding of the target with the probe is detected by a wavelength shift in the refection spectrum.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. application Ser. No.13/303,830 filed Nov. 23, 2011, which is a Continuation of U.S.application Ser. No. 12/923,860 filed Oct. 12, 2010, which is aDivisional of U.S. application Ser. No. 12/662,928 filed May 12, 2010,which is a Divisional of U.S. application Ser. No. 11/699,362 filed Jan.30, 2007. Priority is claimed based on U.S. application Ser. No.13/303,830 filed Nov. 23, 2011, which claims the priority date of U.S.application Ser. No. 12/923,860 filed Oct. 12, 2010, which claims thepriority date of U.S. application Ser. No. 12/662,928 filed May 12,2010, which claims the priority date of U.S. application Ser. No.11/699,362 filed Jan. 30, 2007, which claims the priority date ofJapanese Patent Application No. 2006-061593 filed Mar. 7, 2006, all ofwhich is incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to cells for biochemical analysis, a kitfor biochemical analysis used for detection of a biochemical substance,and a biochemical analyzer.

BACKGROUND OF THE INVENTION

Conventionally, measurement of binding between biochemical substances,such as an antigen-antibody reaction, has been generally performed byusing labels, such as radioactive substances and fluorescent substances.The use of labels needs time. Especially, in using them in a protein,there are a case where the method is complicated and a case whereproperties of a protein will change.

To circumvent these problems, a biochemical sensor that uses a change ofinterference color of an optical thin film is known as a method ofdirectly measuring the binding between biochemical substances withoutusing a label. A paper by T. Sandstrom, et al., Applied Optics, 1985,24, 472-479 (nonpatent document 1) describes this biochemical sensor.Its example will be explained using a model of FIG. 1. An optical thinfilm 1-2 is formed on a substrate 1-1. The refractive index of air is1.00, and the optical thin film 1-2 is a material of a refractive indexof 1.50. The substrate having a refractive index of 2.25 is used. If anoptical thickness of the optical thin film is chosen to be ¼ of avisible light wavelength λ₀ or one of its odd multiples (¾λ₀, 5/4λ₀,etc.), the optical thin film acts as an antireflective film, producingan interference color. On this optical thin film 1-2, a monomolecularlayer of a first biochemical substance 1-3 is formed. If the biochemicalsubstance is considered a protein, its refractive index is of the orderof 1.5 and its layer thickness is of the order of 10 nm. At this time,as shown by a reflection spectrum A of FIG. 2, the intensity ofreflected light in a direction perpendicular to the optical thin filmbecomes zero at wavelength λ₀. When a second biochemical substance 1-4forms bond with this first biochemical substance 1-3 biochemically, achange in the reflection spectrum from a solid line A of FIG. 2 to adashed line A′ occurs, causing the interference color to change. By thischange, binding of the second biochemical substance is detected. As ageneral procedure of detection, first, the optical thin film 1-2 on thesubstrate 1-1 covered with the monomolecular layer 1-3 of the firstbiochemical substance is prepared. This is immersed in a solution of thesecond biochemical substance. Subsequently, it is taken out from thesolution and dried, a change of the interference color from the solidline A of FIG. 2 to the dashed line A′ is examined. Moreover, thedocument describes that the use of a material that is an opticalabsorbing material, for example, silicon, as a material of the substrate1-1 can suppress an effect on the measurement caused by the opticalreflection generated by the back of the substrate. As in the above, thenonpatent document 1 describes a technique wherein, after the sensor istaken out into air and dried, the interference color is measured.

On the other hand, if a material of a refractive index of approximately2.2 is used as the optical thin film, a clear interference color can beobtained in an aqueous solution, and accordingly the amount of bindingof the first biochemical substance and the second biochemical substancecan be measured in real time in the aqueous solution (see a paper by T.Fujimura, et al., Jpn. J. Appl. Phys. 2005, 44, 2849-2853; nonpatentdocument 2). Its example will be explained using a model of FIG. 3. Anoptical thin film 3-2 is formed on a silicon substrate 3-1. The opticalthin film 3-2 is a material of a refractive index of 2.2 and itsthickness is specified to be 70 nm. On this optical thin film 3-2, amonomolecular layer 3-3 of the first biochemical substance is formed.White light is made incident on that structure through an optical window3-4 made of a transparent material, and a reflection spectrum of thesensor is measured. Moreover, if a bundle of optical fiber is used as alight guide for irradiating white light and collecting reflected light,the size of the sensor can be designed to be of a diameter ofsubmillimeter. FIG. 4 shows the reflection spectrum. In calculation ofthe reflection spectrum shown in this FIG. 4, since reflection of thelight on the surface of the optical window 3-4 hardly affects themeasurement, it is ignored. This is done because, by setting aseparation between the optical window 3-4 and the optical thin film 3-2to, for example, approximately 0.15 mm, optical interference between theoptical window 3-4 and the optical thin film 3-2 can be prevented fromaffecting the measurement. The refractive index of a material betweenthe optical window 3-4 and the optical thin film 3-2 was set to arefractive index of water, i.e., 1.333. A layer 3-8 of the firstbiochemical substance and a layer 3-5 of the second biochemicalsubstance are both specified to be a layer of a refractive index 1.5 anda thickness of 10 nm. A solid line B of FIG. 4 shows a reflectionspectrum in the case of absence of the second biochemical substancelayer 3-5; a dashed line B′ of FIG. 4 shows a reflection spectrum in thecase of presence of the second biochemical substance layer 3-5. If thesecond biochemical substance forms bond with the first biochemicalsubstance, a change from the solid line B to the dashed line B′ willoccur and a minimum position of the reflectance will move to a longerwavelength side by 13.5 nm. By measuring this change, the binding of thesecond biochemical substance with the first biochemical substance can bemeasured. Here, in the nonpatent document 1, the refractive index of thelayer of a biochemical substance is set to 1.5, and it can be estimatedthat the layer of a biochemical substance material 3 nm thick within adimensional range of 10 μm×10 μm contains an organic material of 0.5 pg.From the estimate of this nonpatent document 1, a change of the minimumposition of the reflectance of 1 nm in the nonpatent document 2 can beapproximated to the amount of the binding of the biochemical substanceof about 1 ng/mm². Since, by this method, measurement of binding can bedone in real time in a specimen solution, saturation of a reaction canbe found without taking out the sensor from the specimen solution;therefore, it can perform measurement more correctly and more quicklythan the method of the nonpatent document 1.

SUMMARY OF THE INVENTION

Generally, specimen containing a biochemical substance is invaluable.When performing measurement, consumption of a specimen can be made smallby reducing a space between a sensor surface and the optical window,namely the volume of a cell. However, when the separation between thesensor surface and the optical window is made smaller in order to reducethe volume of the cell, it becomes impossible to ignore an effect ofoptical interference between those surfaces facing each other. Moreover,adsorption of the biochemical substance on the optical window also posesa problem. As an example, FIG. 5 shows a model of the case where a layer5-5 of a third biochemical substance adheres to an optical window 5-3.This model assumes the following: An optical thin film 5-2 of athickness of 70 nm and a refractive index of 2.2 is formed on a siliconsubstrate 5-1, an optical window is provided above this, a separationbetween the optical window 5-3 and the optical thin film 5-2 isspecified to be 240 nm, and a space 5-4 therebetween is filled withwater (refractive index 1.333). The layer 5-5 of the third biochemicalsubstance is specified to be a film of a refractive index of 1.5 and athickness of 10 nm. FIG. 6 shows a reflection spectrum in this case. Asolid line C represents a reflection spectrum in the case of absence ofthe layer 5-5 of the third biochemical substance; a dashed line C′represents a reflection spectrum in the case of presence of the layer5-5 of the third biochemical substance. Contrary to the result of FIG.4, FIG. 6 shows that the wavelength position giving a minimum in thereflection spectrum is shifted to a shorter wavelength side byadsorption of the biochemical substance on the optical window 5-3. Notethat generally adsorption of the biochemical substance on the opticalwindow 5-3 is nonspecific. Therefore, this nonspecific adsorption on theoptical window 5-3 becomes a noise in the measurement.

The object of this invention is to solve the above-mentionedconventional technological problem and provide simple a cell forbiochemical analysis, a kit for biochemical analysis, and a biochemicalanalyzer that makes it possible to measure the binding of a biochemicalsubstance with a high throughput and with high sensitivity using a smallamount of a chemical solution.

The cell for biochemical analysis of this invention is specified to be acell that is a gap formed by mutually facing planes of a first substrateand a second substrate disposed close to each other, a probe beingimmobilized on the each of the planes and the gap housing a specimensolution. Then, light is irradiated onto the cell for biochemicalanalysis and a change in a spectrum of the reflected light is detected,whereby binding between the probe and the targeted biochemical materialis detected. The cell for biochemical analysis may have a form of a flowcell.

The biochemical substance being referred to here means a substance thatforms bond with other substance, including not only the substancesprovided in vivo, such as proteins, nucleic acids, lipid andsaccharides, but also exogenous substances each of which forms bond witha molecule in a living body, such as a pharmaceutical substance and anendocrine disrupting chemical substance.

According to this invention, the binding of the biochemical substancethat acts as a target to a probe can be detected with high sensitivityusing a small consumption of the specimen.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram showing a structure of the conventional biochemicalsensor;

FIG. 2 is a diagram showing an interference color change of theconventional biochemical sensor;

FIG. 3 is a diagram showing a structure of the conventional biochemicalsensor;

FIG. 4 is a diagram showing an interference color change of theconventional biochemical sensor;

FIG. 5 is a schematic diagram of a sensor on whose optical window abiochemical substance adsorbs;

FIG. 6 is a diagram showing a change in a reflection spectrum caused byadsorption of the biochemical substance on the optical window;

FIG. 7 is a schematic diagram showing one example of a biochemicalsensor according to this invention;

FIG. 8 is a diagram showing a calculation result of the reflectionspectrum;

FIG. 9 is a diagram showing a relation between the refractive index ofthe layer of a biochemical substance and the wavelength shift;

FIG. 10 is a diagram showing a relation between the separation betweensubstrates and the wavelength shift of the reflection spectrum;

FIG. 11 is a schematic diagram showing other example of the biochemicalsensor according to this invention;

FIG. 12 is a diagram showing a calculation result of the reflectionspectrum;

FIGS. 13A, 13B, and 13C are diagrams showing an example of a productionmethod of a kit for biochemical analysis, in which FIG. 13A showspreparation of first and second substrates, FIG. 13B showsimmobilization of probes on the first and second substrates, and FIG.13C shows fixing of the substrates;

FIG. 14 a schematic diagram showing one example of a detection unit ofthe biochemical analyzer according to this invention;

FIG. 15 is a partial enlarged view of a cell for biochemical analysisbeing set in the biochemical analyzer;

FIG. 16 is a partial enlarged view of the biochemical analyzer;

FIG. 17 is a schematic diagram of one example of a cramp and itssurroundings;

FIGS. 18A, 18B, and 18C are diagrams showing a production method of akit for biochemical analysis in the case where a substrate is specifiedto be made of a resin, in which FIG. 18A shows preparation of the firstand second substrates, FIG. 18B shows immobilization of probes on thefirst and second substrates, and FIG. 18C shows fixing of thesubstrates;

FIGS. 19A, 19B, 19C, and 19D are diagrams showing a production processof a kit for biochemical analysis using a nanoimprint method, in whichFIG. 19A shows a raw material made from polystyrene and a metal moldmade from nickel, FIG. 19B shows pressing of the raw material with themetal mold, FIG. 19C shows a substrate with a member for keepingseparation between the substrates, and FIG. 19D is an enlarged view ofthe member for keeping the separation between the substrates;

FIGS. 20A, 20B, 20C, 20D, 20E, 20F, 20G, and 20H are diagrams showing amethod for manufacturing a metal mold, in which FIG. 20A shows a mastermaterial of silicon wafer, FIG. 20B shows exposure of a photoresist,FIG. 20C shows removal of the photoresist, FIG. 20E shows a metal moldmaster, FIG. 20F shows the width and the period of a wall, FIG. 20Gshows formation of a metal mold, and FIG. 20H shows a metal mold;

FIG. 21 is a diagram showing an example of a member for keeping aseparation between substrates;

FIGS. 22A, 22B, and 22C are diagrams showing a production method of akit for biochemical analysis that constitutes a flow cell, in which FIG.22A shows preparation of the first and the second substrates, FIG. 22Bshows provision of a mask on PDMS, and FIG. 22C shows adhesion of thefirst and second substrates;

FIG. 23 is an enlarged view of an array of slots for keeping theseparation between the substrates;

FIG. 24 is a schematic diagram of a biochemical analyzer using the flowcell;

FIGS. 25A and 25B are diagrams showing a manufacture procedure of asubstrate having an optical thin film, in which FIG. 25A showspreparation of the second substrate, and FIG. 25B shows a sectional viewof FIG. 25A; and

FIGS. 26A and 26B are diagrams showing a method for use of a kit forbiochemical analysis, in which FIG. 26A shows immobilization of probeson the slide glass and the second substrate, and FIG. 26B shows aschematic diagram showing one example of a detection unit of thebiochemical analyzer according to this invention.

DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION

Hereafter, an example of a kit for biochemical analysis and abiochemical analyzer of this invention will be described. As shown inFIG. 7, a first substrate 7-1 and a second substrate 7-2 both made up ofany of glasses, polystyrene, PDMS (polydimethyl siloxane), etc. areprepared. A biochemical substance 7-3 acting as a probe (hereinafterreferred to simply as a probe) is immobilized on surfaces of the firstand second substrates. The surfaces on which the probe are immobilizedare faced each other. A space formed between theses surfaces faced eachother serves as a cell in which a solution containing a specimen isintroduced. Moreover, in order to detect binding of the biochemicalsubstance that is intended to be a target (hereinafter referred tosimply as a target) with the probe, a light source and a detector areprepared. Light from the light source is irradiated from one substrateside, and light reflected from the first substrate and the secondsubstrate is detected with a detector. In this embodiment, the light isirradiated from the first substrate 7-1 side. A specimen solution isintroduced into the cell, and the binding of the target with the probeis detected from an intensity change of the reflected light atwavelengths. In order to prevent the reflected light generated at aplane opposite to a plane of the second substrate 7-2 on which the probeis immobilized from entering the detector to inhibit the measurement,one of the following measures is preferable: using an optical absorbingmaterial as the second substrate 7-2; making a plane opposite to a planeof the second substrate 7-2 on which the probe is immobilizednonparallel thereto so that this reflected light may not immediatelyreturn to the detector; and forming an antireflective coating on a planeopposite to the plane of the second substrate 7-2 on which the probe isimmobilized in order to weaken the intensity of this reflected light.

FIG. 8 shows a calculation result of the reflection spectra in the casewhere the separation between the first substrate 7-1 and the secondsubstrate 7-2 is set to 240 nm, the layer 7-3 of the probe and a layer7-4 of the target are both specified to be a layer having a refractiveindex of 1.5 and a thickness of 10 nm, respectively, and the refractiveindex of a liquid 7-5 between them is set to 1.333. Here, since light isrequired to be incident on the surfaces of the first substrate and thesecond substrate almost vertical thereto and the reflected light isdetected in this geometry, the incident angle of the incident light isset to 0°. In the figure, a solid line D shows a reflection spectrum inthe case of absence of the target layer 7-4 on the both substrate; adashed line D′ shows a reflection spectrum in the case of presence ofthe target layer 7-4 on the both substrates. Since a difference inoptical path length between reflected light beams generated onrespective surfaces of the first substrate 7-1 and the second substrate7-2 facing each other is approximately 600 nm, the minimum in thereflection spectrum appears in the vicinity of a wavelength of 600 nm.The figure shows that a wavelength position giving the minimum in thereflection spectrum shifted to a short wavelength side by the bindingwith the target. This is because reflection of light occurs on thesurface of the respective layers of the biochemical substance added onthe first substrate 7-1 and the second substrate 7-2, not on thesurfaces of the first substrate 7-1 and the second substrate 7-2, andaccordingly the difference in optical path length becomes shorter thanthat in the case of absence of the layers of the biochemical substance,which causes a change in the intensities of the reflected light atwavelengths. The magnitude of the wavelength shift at this time is 53.3nm, being about 4 times the magnitude of the wavelength shift of thenonpatent document 2. Thus, the binding of the target with the probe canbe detected with high sensitivity while the amount of specimen requiredto fill the cell, i.e., the amount of specimen consumed by themeasurement is decreased by reducing the thickness of the cell.

Moreover, gradual variation of the refractive index as well as thedetermination as to whether there is the layer of a biochemicalsubstance of a refractive index of 1.5 can be measured. FIG. 9 shows avariation of a wavelength giving the minimum in the reflection spectrumwhen the refractive index of the target layer is varied from 1.333 to1.5. It can be seen from the figure that the wavelength position givingthe minimum in the reflection spectrum varies almost linearly tovariation of the refractive index. Here, this gradual change of therefractive index corresponds to a gradual change of the density of thebinding of the target. This correspondence relation can be explained,for example, by an effective medium approximation of the Lorentz-Lorenztheory that is described in a paper by M. Harris, et al., Thin SolidFilms, 1979, 57, 173-178, or the like. From the foregoing, the gradualchange of the density of the binding of a biochemical substance can bemeasured as a change of the wavelength shift.

On the other hand, assumption of the thickness of the layer of abiochemical substance is done corresponding to the size of thebiochemical substance that is considered. The thickness of the layer ofthe biochemical substance was assumed 10 nm in this embodiment. Themagnitude of the wavelength shift is in proportion to the layerthickness of this biochemical substance. For example, if a smallerbiochemical substance is considered and the thickness of the layer of abiochemical substance is assumed to be 1 nm, the magnitude of thewavelength shift will become 1/10 of a value when assuming the thicknessto be 10 nm.

In the case where the separation between the substrates is specified tobe 240 nm, the minimum in the reflection spectrum that appears in thevicinity of a wavelength of 600 nm is caused by first-order opticalinterference. Thus, for improvement in detection sensitivity, it isdesirable to set the separation between the first and second substratesto a separation where the minimum in the reflection spectrum bylow-order optical interference appears in a wavelength band of lightused for the measurement. The reason will be explained below.

Since when the separation between the substrates is set to two times 240nm, i.e., 480 nm, a difference in optical path length between thereflected light beams generated on respective surfaces of the firstsubstrate 7-1 and the second substrate 7-2 facing each other isapproximately 1200 nm, the minimum in the reflection spectrum appears inthe vicinity of a wavelength of 600 nm by second-order opticalinterference. When the separation between the substrates is set to threetimes 240 nm, i.e., 720 nm, the minimum in the reflection spectrumappears in the vicinity of a wavelength of 600 nm by third-order opticalinterference. FIG. 10 show a plot of a relation of the magnitude of thewavelength shift of a minimum position in the reflection spectrum in thevicinity of a wavelength of approximately 600 nm obtained when thetarget forms bond with the probe versus the magnitude of the separationbetween the first and second substrates. Here, the layers of thebiochemical substance that was targeted were assumed to have arefractive index of 1.5 and a thickness of 10 nm. As can be understoodfrom FIG. 10, the magnitude of the wavelength shift of the minimumposition in the reflection spectrum obtained when the target forms bondwith the probe is in inverse proportion to the magnitude of theseparation between the first and second substrates. For example, whenthis separation between the substrates is 240 nm, the magnitude of thiswavelength shift was 53.3 nm, while when this separation between thesubstrates is increased to a double, i.e., 480 nm, the magnitude of thiswavelength shift becomes a half, i.e., 26.7 nm. If the order n ofoptical interference further increases, the magnitude of the wavelengthshift will become one n-th. From the above, using optical interface of alow order is advantageous for higher sensitivity.

In addition, even when the optical thin film is formed on one of thesubstrates, as shown in FIG. 5, high-sensitivity measurement isrealizable by immobilizing the probe not only on the surface of theoptical thin film but also on the surface of the optical window andbringing the optical thin film closer to the optical window than theforegoing case. As shown in FIG. 11, an optical thin film 11-2 of athickness of 70 nm and a refractive index of 2.2 is formed on a siliconsubstrate 11-1. By the presence of this optical thin film 11-2, theminimum in the reflection spectrum appears in the vicinity of awavelength of 600 nm that corresponds to four times the opticalthickness. An optical window 11-3 is formed above this optical thin film11-2. The separation from the surface of this optical thin film 11-2 tothe optical window 11-3 is set to 50 nm. Moreover, probes are providedon the surface of the optical thin film 11-2 and the surface of theoptical window 11-3, respectively. FIG. 12 show a calculation result ofthe reflection spectrum in the case where layers 11-4 of the probe andlayers 11-5 of the target are specified to have each a refractive indexof 1.5 and a thickness of 10 nm and a liquid 11-6 therebetween isspecified to have a refractive index of 1.333. A solid line E of FIG. 12represents a reflection spectrum in the case of absence of the layer11-5 of the target; a dashed line E of FIG. 11 represents a reflectionspectrum in the case of presence of the layer 11-5 of the target. Thegraphs show that the biding of the biochemical substance shifts awavelength position giving the minimum in the reflection spectrum to along wavelength side. The magnitude of this wavelength shift is 23.1 nm,which is 1.7 times the magnitude of a shift of the nonpatent document 2.This increase of the signal is attributed to a fact that addition of thelayer 11-5 of the target on the optical window 11-3 contributes to thewavelength shift because of setting the separation between the surfaceof the optical thin film 11-2 and the optical window 11-3 to 50 nm.Thus, the binding of the target with the probe can be detected with highsensitivity. Note that it is preferable that the separation between thesubstrate of the optical thin film and the substrate of the opticalwindow is 10 nm or more considering the size of a protein that is theprobe or a protein that is targeted.

As described above, the binding of the target with the probe can bedetected with high sensitivity without using a label in a state wherethe probe is immobilized on the surfaces of the both substrates, therebydecreasing the separation between the substrates, i.e., in a state wherethe volume of the cell is made smaller.

DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION First Embodiment

Hereafter, an embodiment of a simple cell for biochemical analysis and asimple biochemical analyzer of this invention that uses opticalinterference will be described.

First, a production method of a kit for biochemical analysis will beexplained. FIG. 13 shows a production method of the kit for biochemicalanalysis. As shown in FIG. 13A, a first substrate 13-1 and a secondsubstrate 13-2 having a flat plane are prepared. The first substrate13-1 and the second substrate 13-2 can be made of a transparent glass. Avertical elevation 13-3 of approximately 240-nm height is formed in thefirst substrate 13-1, and a flat plane 13-5 is made higher by 240 nmthan a flat plane 13-4 beforehand. The second substrate 13-2 is providedwith a plane 13-6 nonparallel to the flat plane on the opposite sidethereof beforehand. The flat plane 13-4 of the first substrate 13-1 andthe flat surface of the second substrate are silane-coated using3-aminopropyltrimethoxysilane. Since the surface subjected to thisprocessing is hydrophilic, an aqueous solution can be introduced into acell formed between the first substrate and the second substrate bycapillarity. Moreover, a biochemical substance can be immobilized usingan amino group introduced by this processing. For example, in order toimmobilize a protein as the probe, a protein carboxyl group and an aminogroup introduced on the surface can be brought into amide binding usingan aqueous solution of N-hydroxysucciimide and water solublecarbodiimide. Alternatively, performing silane coating on the flat plane13-4 of the first substrate 13-1 and the flat surface of the secondsubstrate 13-2 using 3-glycidoxypropyltrimethoxysilane also makes itpossible to obtain hydrophilic surfaces similarly. Moreover, using anepoxy group introduced by this processing, a biochemical substance canbe immobilized by dehydrating condensation with the amino group or ahydroxyl group.

Note that a pair of the first substrate 13-1 and the second substrate13-2 with probes immobilized thereon may be offered as a kit forbiochemical analysis, or such a pair with probes not immobilized thereonmay be offered as a kit for biochemical analysis, leaving immobilizationof the probes to the user.

Next, as shown in FIG. 13B, probes 13-7, 13-8, 13-9, and 13-10 areimmobilized on the flat plane 13-4 of the first substrate 13-1 and theflat surface of the second substrate 13-2. At this time, positions atwhich the probes are immobilized are so determined that, when thesubstrates are faced each other, the sites on which the same kind ofprobe is immobilized face each other. Moreover, at this time, the probeis intended to be immobilized on the opposite side of the nonparallelplane 13-6 in the substrate 13-2. After the probe is immobilized, asshown in FIG. 13C, the planes on which the probes are immobilized arefaced each other, and the substrates are fixed to each other with acramp etc. in a state where the flat plane 13-5 of the first substrate13-1 and the flat plane of the second substrate 13-2 contact with eachother. By this process, a gap with a vertical elevation 13-3 in a heightdirection is formed between the planes on which the probe isimmobilized, respectively, and thus the cell can be formed.

One example of a procedure of detecting the binding of the target withthe probe of this kit for biochemical analysis will be shown below. FIG.14 is a schematic diagram showing one example of a detection unit of abiochemical analyzer according to this invention. In order to introducea specimen solution into the cell formed between the first substrate13-1 and the second substrate 13-2 that are fixed with a clamp etc., amechanical pipet 14-1 is used to drop the specimen solution near thecell. The specimen solution dropped near the cell is introduced into thecell by capillarity. The binding of the target introduced into the celland the probe immobilized on the surfaces of the first substrate 13-1and the second substrate 13-2 is measured by an optical detection systembelow. Using a light guide 14-2 made up of an optical fiber bundle,light from a white light source 14-3 is irradiated from the firstsubstrate 13-1 side, and the reflected light from the surfaces of thefirst substrate 13-1 and the second substrate 13-2 on each of which theprobe is immobilized is guided to respective spectrometers 14-4. Acomputer 14-5 captures each reflection spectrum in real time, calculatesa wavelength giving a minimum in each reflection spectrum, and performsdisplay and recording of its temporal change in real time.

FIG. 15 is a partial enlarged view of the biochemical sensor installedin the biochemical analyzer, showing a sectional view of the light guide14-2, a stage 15-3 on which the light guide 14-2 is fixed, the firstsubstrate 13-1, and the second substrate 13-2. The light guide 14-2consists of an optical fiber bundle in which strands of optical fiber15-1 to guide the light from the light source are tied to surroundoptical fiber 15-2 to guide the light to the spectrometer. As shown inFIG. 15, the point of the light guide 14-2 and the first substrate 13-1are arranged to almost touch each other. This disposition can preventthe reflected light from the back of the first substrate from directlyentering the optical fiber 15-2 for guiding the reflected light to thespectrometer. Moreover, since the nonparallel plane 13-6 that is anupper plane of the second substrate 13-2 is inclined, the reflectedlight from the nonparallel plane 13-6 can be prevented from directlyreturning to the optical fiber 15-1. Alternatively, the same effect canbe obtained by placing an antireflective coating on the plane 13-6 toprevent the reflection itself of the light on the plane 13-6. Furtheralternatively, by specifying the substrate 13-2 to be made of an opticalabsorbing glass, such as a black glass, the transmitted light in thesubstrate 13-2 that is a part of the light irradiated from the opticalfiber 15-2 is made to be absorbed in the substrate 13-2. Therefore, theeffect on the measurement caused by the transmitted light entering theoptical fiber 15-2 for guiding the reflected light to the spectrometerafter being reflected on the interface can be avoided. Moreover, byspecifying the substrate 13-2 to be made of a black glass, the effect onthe measurement caused by the indoor light entering the optical fiber15-2 for guiding the reflected light to the spectrometer can be avoided.

FIG. 16 shows one example of the biochemical analyzer for performing theabove measurement. The biochemical analyzer is provided with a black box16-1 for housing the first substrate 13-1, the second substrate 13-2,the mechanical pipet 14-1, a point of the light guide 14-2, and thestage 15-3 in it. By using this black box 16-1, the effect on themeasurement caused by the indoor light entering the optical fiber 15-2for guiding the reflected light to the spectrometer can be avoided. Theblack box 16-1 is provided with a lid 16-2. Setup is done with the lid16-2 opened and the measurement is performed with the lid 16-2 closed.The black box is further provided with a clamp for fixing the firstsubstrate 13-1 and the second substrate 13-2 in it.

FIG. 17 is a schematic diagram of one example of the clamp and itssurroundings. The illustrated clamp fixes the first substrate 13-1 andthe second substrate 13-2 by sandwiching them with the stage 15-3 and aboard 17-1, and pressing down the board 17-1 with screws. A groove 17-2into which the first substrate 13-1 and the second substrate 13-2 issettable is formed in the stage 15-3. The form of the groove 17-2 shallbe such that its width just accommodates the first substrate 13-1 andits depth houses the whole of the first substrate 13-1 and a part of thesecond substrate 13-2, the first substrate 13-1 and the second substrate13-2 being combined with each other. Moreover, the position of thisgroove 17-2 is so adjusted that the points of the light guide 14-2 comeexactly under the positions at which the probes 13-7, 13-8, 13-9, and13-10 are fixed. The light source 14-3, points of the light guide 14-2on the light source side and on the spectrometer side, the spectrometers14-4, and the computer 14-5 are placed and used outside the black box.

Second Embodiment

Although the substrate is specified to be made of a glass in the firstembodiment, the kit for biochemical analysis can be produced withresins, such as polystyrene and PDMS. FIG. 18 shows a production methodof a kit for biochemical analysis in the case where a resin is used fora substrate. As shown in FIG. 18A, a first substrate 18-1 and a secondsubstrate 18-2 having a flat plane are prepared. In this embodiment, thesubstrate 18-1 and the substrate 18-2 are specified to be made oftransparent polystyrene. As shown in FIG. 18A, the first substrate 18-1is provided with a vertical elevation 18-3 of 240-nm height and a member18-4 for keeping the separation between the first substrate 18-1 inwhich a wall of approximately 240-nm height and 80-nm width are arrangedin a period of 400 nm and the other substrate. Note that, with the helpof the vertical elevation 18-3, a flat plane 18-5 of the substrate 18-1is made higher than the other flat plane 18-6 by 240 nm. Like the firstembodiment, a plane 18-7 nonparallel to the flat plane and opposite toit is provided on the second substrate 18-2.

The vertical elevation of the first substrate 18-1 and the member 18-4for keeping the separation can be manufactured by a method that will bedescribed below. FIG. 19 is a production process diagram of a kit forbiochemical analysis using a nanoimprint method in this embodiment. Asshown in FIG. 19A, a raw material 19-1 made from polystyrene (26 mm×40mm, and 1 mm in thickness) and a metal mold 19-2 made from nickel areprepared. As shown in FIG. 19B, this raw material 19-1 and thenickel-made metal mold 19-2 heated to 150° C. are pressed for 10 secondswith a press pressure of 25 MPa. Then, by separating the metal mold 19-2from the substrate 19-1 perpendicularly, the substrate 18-1 that has thevertical elevation 18-3 of 240-nm height and the member 18-4 for keepingthe separation between the substrates can be obtained, as shown in FIG.19C. FIG. 19D shows an enlarged view of the member 18-4 for keeping theseparation between the substrates. The height F of a wall isapproximately 240 nm, the width G of the wall is 80 nm, and the period Hof its array is 400 nm. Here, an arrow 19-3 of FIG. 19C and an arrow19-4 of FIG. 19D show the same direction.

FIG. 20 shows a method for manufacturing the metal mold 19-2. A mastermaterial 20-1 shown in FIG. 20A is a silicon wafer of a crystalorientation (100) and dimensions of 26 mm×40 mm. As shown in FIG. 20B, aphotoresist 20-2 is applied on the master material 20-1 and thephotoresist (resist) located at the vertical elevation and the wall issubjected to exposure by an electron beam writing system. Subsequently,as shown in FIG. 20C, the resist of an exposed portion is removed by adevelopment process. In a photoresist remaining portion 20-3 shown bythe hatched area of FIG. 20C, the resist in the whole hatched area isremained, while in a resist remaining portion 20-4 shown by anotherhatched area, the resist is remained partly as in the form of a grid.FIG. 20D is an enlarged view of a grid-like pattern in the portion 20-4where the resist remains. The width and the period of a grid 20-6 ofthis resist are the same as the values of G and H shown in FIG. 19D. Anarrow 20-7 shows the same direction as an arrow 20-5 of FIG. 20C. Next,as shown in FIG. 20E, dry etching is used to form a metal mold master20-8 in which a shape corresponding to the vertical elevation and thewall is formed. FIG. 20F is an enlarged view of an area 20-9 in whichthe array of walls formed by dry etching. The width and the period of awall 20-11 of FIG. 20F are the same as G and H shown in FIG. 20D. Anarrow 20-12 shows the same direction as an arrow 20-10 of FIG. 20E.Subsequently, as shown in FIG. 20G, a nickel thin film is formed on themetal mold master 20-8 by an electroless plating method, and then thenickel thickness is increased to 1 mm by an electrolytic plating methodto form a metal mold 20-13. Subsequently, by performing separationprocessing with a predetermined fluorinated agent on the surface of themetal mold 20-13, a metal mold 20-16 that has a mold 20-14 of thevertical elevation of 240-nm height shown in FIG. 20H and a mold 20-15of the member for keeping the separation between the substrates can beobtained.

Although in this embodiment, the vertical elevation and the array ofwalls were formed using polystyrene as a raw material by a nanoimprintmethod, other molding methods, including the cast method of pouring aliquid material onto a metal mold, can also be used. Especially whenPDMS that is transparent and adheres to the substrate is used, it ispreferable to use the cast method. Moreover, although in thisembodiment, the member 18-4 for keeping the separation between thesubstrates was specified to be the array of walls, members of othershapes, such as of an array of cylinders 21-1 as shown in FIG. 21, maybe used.

Returning to FIG. 18, after preparing the first substrate 18-1 and thesecond substrate 18-2, the area 18-4 of the first substrate 18-1 and theflat plane of the second substrate 18-2 are silane-coated using3-aminopropyltrimethoxysilane. Since the surface subjected to thisprocessing is hydrophilic, an aqueous solution can be introduced into acell formed in a gap between the first substrate and the secondsubstrate by capillarity. Moreover, a biochemical substance (probe) canbe immobilized using an amino group introduced by this processing.Alternatively, like the first embodiment, a hydrophilic surface isobtained by silane-coating the surface of the substrate using3-glycidoxypropyltrimethoxysilane, whereby, as well as by the use of anepoxy group introduced by this process, a biochemical substance can beimmobilized. Then, as shown in FIG. 18B, probes 18-8, 18-9, 18-10, and18-11 are immobilized on the area 18-4 of the first substrate 18-1 andthe surface of the second substrate 18-2. At this time, positions atwhich the probes are immobilized are so determined that the sites onwhich the same kind of probe is immobilized face each other when thesubstrates are faced each other. After the probe is immobilized, theplanes on which the probe is immobilized are faced each other, as shownin FIG. 18C, and the substrates are fixed with a cramp like the firstembodiment. By this fixing, a cell whose height is kept by the memberfor keeping the separation between the substrates can be formed betweenthe planes on which the probe is immobilized, respectively.Incidentally, in the case where a material having a self-adhesionproperty is used for either the first substrate 18-1 or the secondsubstrate 18-2, or used for the both the first substrate 18-1 and thesecond substrate 18-2, a state where the first substrate 18-1 keepsadhering to the second substrate 18-2 can be maintained without usingthe cramp.

Note that a pair of the first substrate 18-1 and the second substrate18-2 with probes immobilized thereon may be offered as a kit forbiochemical analysis, or such a pair with probes not immobilized thereonmay be offered as a kit for biochemical analysis, leaving immobilizationof the probes to the user.

Detection of the binding of the target with the probe of this cell forbiochemical analysis is possible by using the same biochemical analyzerand the same measurement procedure as those of the first embodiment.Moreover, like the first embodiment, by specifying the second substrate18-2 to be black-colored, the light transmitted in the second substrate18-2 that is a part of the light irradiated from the optical fiber 15-1is absorbed in the second substrate 18-2, which can avoid the effect onthe measurement caused by the transmitted light entering the opticalfiber 15-2 for guiding the reflected light to the spectrometer afterbeing reflected on the interface. Furthermore, by specifying the secondsubstrate 18-2 to be light absorbing, such as of black color, the effecton the measurement caused by the indoor light entering the optical fiber15-2 for guiding the reflected light to spectrometer can be avoidedwithout using the black box.

Third Embodiment

In the first embodiment and the second embodiment, the specimen solutionis introduced into the cell using capillarity. The binding of the targetwith the probe can also be measured as follows: A space formed betweenthe substrates is used as a flow cell and a state where the specimensolution flows in the cell regularly is made, for example, by applying apressure to it. This embodiment explains an example of the case where aspace formed between the substrates is used as the flow cell.

FIG. 22 is an explanatory diagram about a production method of a kit forbiochemical analysis in the case where a gap for holding a specimensolution is specified to be the flow cell. As shown in FIG. 22A, a firstsubstrate 22-1 and a second substrate 22-2 having a flat plane areprepared. The first substrate 22-1 and the second substrate 22-2 arespecified to be made of PDMS. As shown in FIG. 22A, the first substrate22-1 is provided beforehand with an area 22-3 having an array of slotsthat is intended to form a gap between the substrates and is obtained byarranging slots each having a depth of 240 nm and a width of 320 nm in aperiod of 400 nm. FIG. 23 is an enlarged view the array of slots forkeeping this separation between the substrates. The depth J of thegroove is approximately 240 nm, the width K of the groove is 320 nm, andthe period L of its array is 400 nm. Here, an arrow 22-4 of FIG. 22 andan arrow 23-1 of FIG. 23 show the same direction. On the other hand,holes 22-5, 22-6 each for allowing a tube for applying a pullingpressure to the array of slots to be connected are formed beforehand inthe second substrate 22-2. Moreover, like the first embodiment and thesecond embodiment, on the opposite side of the flat plane of the secondsubstrate 22-2, a plane 22-7 nonparallel to the flat plane to avoid theeffect of the reflected light is provided beforehand.

Incidentally, it is not necessarily required to form the array of slotsin the area 22-3 of the first substrate. In the case where the array ofslots is not formed in the area 22-3, the area 22-3 becomes a flat planedepressed from a surrounding plane by approximately 240 nm.

A mask 22-8 is given by sticking a film of a resin that has a propertyof self-adhesion to PDMS, as shown in FIG. 22B, after preparing thefirst substrate 22-1 and the second substrate 22-2. The area 22-3 of thefirst substrate 22-1 in which the slots are formed and a part of theflat plane of the second substrate that is not masked are subjected tosilane-coating using 3-aminopropyltrimethoxysilane. Since the surfacesubjected to this processing is hydrophilic, an aqueous solution can beintroduced into a cell formed between the first substrate and the secondsubstrate by capillarity. Moreover, a biochemical substance (probe) canbe immobilized using an amino group introduced by this processing.Alternatively, like the first embodiment, a hydrophilic surface isobtained by silane-coating the surface of the substrate using3-glycidoxypropyltrimethoxysilane, whereby, as well as by the use of anepoxy group introduced by this process, a biochemical substance can beimmobilized. Then, as shown in FIG. 22B, probes 22-9, 22-10, 22-11, and22-12 are immobilized on the area 22-3 of the first substrate 22-1 inwhich the array of slots is formed and on the surface of the flat planeof the second substrate 22-2. At this time, positions at which theprobes are immobilized are so determined that the sites on which thesame kind of probe is immobilized face each other when the substratesare faced each other. Then, the mask 22-8 is removed from the firstsubstrate 22-1 and the second substrate 22-2, and the first substrate22-1 and the second substrate 22-2 are adhered using their property ofself-adhesion, as shown by FIG. 22C.

Note that a pair of the first substrate 22-1 and the second substrate22-2 with probes immobilized thereon may be offered as a kit forbiochemical analysis, or such a pair with probes not immobilized thereonmay be offered as a kit for biochemical analysis, leaving immobilizationof the probes to the user.

FIG. 24 schematically shows an analyzer and a method for detecting thebinding of a target with a probe using this kit of biochemical analysis.A tube 24-3 is connected to the hole 22-6 of the second substrate 22-2for allowing a tube to be connected. A syringe 24-4 is connected to theopposite side of this tube 24-3. Using a syringe pump 24-5, the syringeis evacuated to apply a pulling pressure to the flow cell. A tube 24-6is connected to the hole 22-5 of the second substrate for allowinganother tube to be connected. A valve 24-7 is connected to the oppositeside of this tube 24-6. By switching this valve 24-7, a state of sendinga buffer solution 24-9 can be changed to a state of sending a specimensolution 24-8.

Moreover, plural valves are attached to the analyzer, as shown in FIG.24. Furthermore, a dilute hydrochloric acid is put in one of thecontainers connected to the valve, and is injected into the flow cellfor three minutes, whereby a biochemical substance (target) formed bondwith the probe when each specimen solution is flown in the flow cell canbe dissociated from the biochemical substance immobilized on thesubstrate as a probe. By this configuration, different specimensolutions can be injected into the flow cell continuously to bemeasured, as follows. First, a state of sending the buffer solution isswitched to a state of sending the specimen solution. The buffersolution is sent again, and a shift of the minimum position in thereflection spectrum with respect to that of the initial state of sendingthe buffer solution is checked. Then, after dissociating the biochemicalsubstance by sending the dilute hydrochloric acid for three minutes, thebuffer solution is sent to return the system to its initial state. Theabove is defined as one cycle. Similarly next injection of a specimensolution can be performed.

The binding of the target in the specimen solution sent into the cellwith the probes immobilized on the surfaces of the first substrate 22-1and the second substrate 22-2 can be measured by the same opticaldetection system as that of the first embodiment. Using the light guide14-2, the light from the white light source 14-3 is irradiated from thefirst substrate 22-1 side, and the reflected light from the surfaces ofthe first substrate 22-1 and the second substrate 22-2 on which theprobes are immobilized is guided to the respective spectrometers 14-4.The computer 14-5 captures the reflection spectrum obtained from theeach spectrometer 14-4 in real time, and reads changes of wavelengthsgiving minimums of the respective reflection spectra.

Like the first embodiment, a point of the light guide 14-2 is disposedto almost touch the first substrate 22-1. By this arrangement, thereflected light from the back of the first substrate is prevented fromdirectly entering the optical fiber 15-2 for guiding the reflected lightto the spectrometer. Moreover, since the nonparallel plane 22-7 of thesecond substrate 22-2 is inclined, the reflected light from thenonparallel plane 22-7 can be prevented from directly returning to theoptical fiber 15-2. In the above measurement, the use of a black box24-1 can avoid the effect on the measurement caused by the indoor lightentering the optical fiber 15-2. The black box 24-1 is provided with alid 24-2 and houses the light guide 14-2, the first substrate 22-1, thesecond substrate 22-2, the tube 24-3, and the tube 24-6. Setup is donewith the lid 24-2 opened, and the measurement is performed with the lid24-2 closed.

Note that if the second substrate is specified to be made of black PDMS,light that is transmitted in the second substrate 22-2 in the portion ofthe light irradiated from the optical fiber 15-1 is allowed to beabsorbed in the substrate 22-2. By this scheme, an effect on themeasurement caused by the transmitted light entering the optical fiber15-2 for guiding the reflected light to the spectrometer after beingreflected on an interface can be avoided. Moreover, by specifying thesecond substrate 22-2 to be made of the black PDMS, the effect on themeasurement caused by indoor light entering the optical fiber 15-2 forguiding the reflected light to the spectrometer can be avoided withoutusing the black box. Although the array of the slots shown in FIG. 23was formed to form the cell in this embodiment, the following is alsopossible: Slits are formed in the area 22-3 of the first substrate 22-1,leaving an array of cylinders as shown in FIG. 21, and the firstsubstrate thus formed is stuck to the second substrate 22-2 to shape acell whose thickness is defined by the height of the cylinders.Moreover, although the probe and the light guide were arranged in a linein a solution sending direction in this embodiment, this may be changedto two or more lines to form a two-dimensional array.

Fourth Embodiment

As described above, by forming an optical thin film whose opticalthickness is ¼ of a wavelength of a visible light or its odd multiple ona single side of the substrate, it is possible to further reduce thedimension of the gap between the substrates than those of the first,second, and third embodiments, and decrease the capacity of the cell. Anembodiment in the case where the thickness of the cell is furtherreduced by using an optical thin film will be explained below.

FIG. 25 is a diagram showing a manufacture procedure of the secondsubstrate in the case of forming an optical thin film on the secondsubstrate. As shown in FIG. 25A, a second substrate 25-1 on which asilicon-nitride thin film whose refractive index is adjusted to 2.2 isformed on a flat surface of a silicon substrate of 26 mm×10 mm isprepared. A silicon-nitride thin film is also formed on its back. Withthis film, chemical resistance of this substrate against alkalinesolutions is improved. The vertical elevation 25-2 of 50-nm height isformed in this second substrate 25-1, and a flat plane 25-4 is madehigher than a flat plane 25-3 by 50 nm beforehand. FIG. 25B shows asectional view taken along the line M-M′ of FIG. 25A. A silicon nitridefilm 25-5 is formed on the back of the silicon substrate. On the surfaceof the silicon substrate, a silicon nitride film 25-6 is formed. Thereis a vertical elevation 25-2 of 50-nm height in the silicon nitride film25-6. A low plane defined by the vertical elevation 25-2 as a boundaryis the flat plane 25-3, and a high plane defined similarly is a flatplane 25-4.

FIG. 26 shows a method for using a sensor kit. Slide glass 26-1 is usedas the first substrate. The slide glass 26-1 and the flat plane 25-3 ofthe second substrate 25-1 are silane-coated using3-aminopropyltrimethoxysilane. Since the surface subjected to thisprocessing is hydrophilic, an aqueous solution can be introduced into acell made in the gap between the first substrate and the secondsubstrate by capillarity. Moreover, a biochemical substance can beimmobilized using an amino group introduced by this processing.Alternatively, like the first embodiment, a hydrophilic surface isobtained by silane-coating the surface of the substrate using3-glycidoxypropyltrimethoxysilane, whereby, as well as by the use of anepoxy group introduced by this process, a biochemical substance can beimmobilized.

Subsequently, as shown in FIG. 26A, probes 26-2, 26-3, 26-4, and 26-5are immobilized on the slide glass 26-1 and the flat plane 25-3 of thesubstrate 25-1. At this time, positions at which the probes areimmobilized are so determined that the sites on which the same kind ofprobe is immobilized face each other when the substrates are faced eachother. After the probe is immobilized, as shown in FIG. 26B, the planeson which the probes are immobilized are faced each other, and thesubstrates are fixed to each other with a cramp like the firstembodiment. By this procedure, a gap with a vertical elevation 25-2 inthe height direction is formed between the planes on which the probesare immobilized, respectively, and thus the cell can be formed.

It is possible to detect the binding of a target with the probe of thissensor kit by using the biochemical analyzer and the same measurementprocedure as those of the first embodiment. At this time, since thesecond substrate 25-1 is opaque, the same effect as in the case wherethe second substrate in the first embodiment is specified to be anoptical absorbing glass, such as of black color, that is, thetransmitted light into the second substrate and indoor light can beprevented from affecting the measurement. Note that the example offorming the optical thin film of a refractive index of 2.2 was explainedin this embodiment. However, the optical thin film may be formed on thefirst substrate instead.

What is claimed is:
 1. A biochemical analyzer, comprising: a cell forbiochemical analysis that has a first substrate having a surface with aflat first plane and second plane, the second plane having a verticalelevation compared to the first plane and second substrate having a flatbase, wherein the second plane of the first substrate is in contact withthe base of the second substrate, and same kind of biochemicalsubstances immobilized on a site of the first plane of the firstsubstrate and a site of the base of the second substrate; a fixingsection for fixing the cell for biochemical analysis; a light source; aspectrometer; an optical system for guiding light from the light sourceto the site of the first plane of the first substrate and the site ofthe base of the second substrate; an optical system for guidingreflected light from the cell for biochemical analysis to thespectrometer; and a computer unit for detecting a change in a reflectionspectrum form an output of the spectrometer.
 2. The biochemical analyzeraccording to claim 1, wherein the fixing section is disposed in a blackbox.